t'eterinary Microbiology, 28 ( 1991 ) 289-294 Elsevier Science Publishers B.V., A m s t e r d a m
289
Dot immunobinding assay for the detection of bluetongue virus antibodies in sheep experimentally inoculated with bluetongue virus type 1 S. Chander, G. Prasad and N.C. Jain Department of Veterinary Microbiology, College Qf Veterinary Sciences, Ha~vana Agricultural University, Hisar-125004, India (Accepted 30 October 1990 )
ABSTRACT Chander, S., Prasad, G. and Jain, N.C., 1991. Dot immunobinding assay for the detection of bluetongue virus antibodies in sheep experimentally inoculated with bluetongue virus type 1. Vet. Microbiol., 28: 289-294. Dot immunobinding assay (D1A) was evaluated for the detection of bluetongue virus (BTV) antibodies in sheep experimentally inoculated with BTV 1. Serum samples collected on 14, 21, 28, 43 and 60 day post infection (dpi) were positive for precipitating antibodies by the agar gel precipitation test (AGPT) while antibodies could be detected as early as 7 dpi by DIA and ELISA. Virus neutralizing antibodies were detected first at 14 dpi. The sensitivity of the four tests was compared on the same serum samples collected at different intervals. The results indicated that DIA was more sensitive than AGPT and the serum neutralization test and as sensitive as ELISA. Thus due to sensitivity simplicity and economy, DIA could replace AGPT for diagnosis and serological survey for BTV infection in animals.
INTRODUCTION
Bluetongue is an insect transmitted viral disease of sheep, goat, cattle and other ruminants. It is caused by bluetongue virus (BTV) which is the prototype orbivirt~s of the family Reoviridae (Murphy et al., 1971 ). Currently the agar gel precipitation test (AGPT) is being used all over the world for screening the animals for BTV antibodies. Reliability of AGPT has been questioned by several workers (Manning and Chen, 1980; Osburn et al., 1981; Stott et al., 1982 ). Enzyme-linked immunosorbent assay (ELISA) has been used during the last decade for detection of group specific BTV antibodies and is comparatively more sensitive than AGPT (Hubschle et al., 1981; Poli et al., 1982; Gupta et al., 1990). Since the ELISA requires sophisticated equipment for
290
s. CHANDER ETAL.
reading the results, dot immunobinding assay (DIA), a modification of ELISA on nitrocellulose membranes, has been developed (Hawkes, 1986; Afshar et al., 1987a; Gupta et al., 1990). In the present study DIA was evaluated for the detection of BTV antibodies in experimentally inoculated sheep. The BTV antibodies were also determined by AGPT, SNT and ELISA for comparative evaluation. MATERIALS AND METHODS
Cell culture Baby hamster kidney (BHK-21 ) cell line, obtained from the Indian Veterinary Research Institute, Mukteshwar, was grown in tissue culture bottles a n d / o r 15 × 125 m m test tubes following the method of Stocker and Macpherson (1961). Growth m e d i u m contained BHK-21 m e d i u m (Glasgow modification) supplemented with 10% inactivated precolostral calf serum and antibodies (penicillin 100 I U / m l , streptomycin sulfate 100 j~g/ml). VirblS
Bluetongue virus serotype 1 available at our laboratory was used to infect the sheep. Animals The 1-year-old crossbred sheep (Nali × Corriedale) free from BTV antibodies were randomly divided in to two groups. Group 1 (seven sheep) was injected with 10 ml of 10 6 TCIDs0/ml of BTV per animal intradermally and subcutaneously at multiple sites. Group 2 (three sheep) were intact, uninfected control sheep. All animals were kept in a midge-proof house. Serum samples Serum samples were collected at 0, 7, 14, 21, 28, 43 and 60 days post infection (dpi) for the detection of BTV antibodies. Serological tests Agar gel precipitation test (AGPT) was carried out following the method of Jochim and Chow ( 1969 ) using standard group specific BTV antigen supplied by Dr. M.M. Jochim, Director, Veterinary Diagnostic Technology Inc. Wheat Ridge, USA. Serum neutralization test (SNT) Neutralizing antibodies against BTV were detected with the method of Howell (1960). The reciprocal of highest serum dilution showing 50% inhibition of cytopathic effect (CPE) was taken as titer of neutralizing antibodies.
DIA FOR BLUETONGUE VIRUS ANTIBODIES IN SHEEP
291
Elisa and dia Substrate. O-phenylenediamine (Sigma, USA) was used as substrate in ELISA, while diaminobenzidine tetrahydrochloride (Sigma, USA) was used in DIA as described by Gupta et al. (1990). Conjugate. Horse radish peroxidase conjugated to rabbit anti-sheep immunoglobulins (DAKOPAT, Denmark) were used at 1:2000 dilution in both tests as described by Gupta et al. (1990). Antigen and antiserum. BTV antigen and antiserum were the same as used for AGPT (Bluetongue antibody test kit, Veterinary Diagnostic Technology, Inc. Wheat Ridge, USA). Indirect ELISA The standard BTV antigen was diluted in carbonate-bicarbonate buffer (pH 9.6) to an OD28o of 0.01 for coating ELISA plates. All other steps of the test were performed as described by Gupta (1988). Absorbance was read at 492 nm wavelength by an ELISA reader (Titertech, Multiscan, Flow Lab.). The end point value of OD was calculated as described by Hubschle et al. ( 1981 ). An OD above 0.3 was taken as positive while an OD equal to or less than 0.3 was considered negative. Dot immunobinding assay Nitrocellulose membranes bound to plastic strips (East Punjab Mfg. Co. Hisar, India) were used for DIA. The test was performed using the protocol developed in our laboratory (Gupta et al., 1990). Briefly, 1 ~tl of the standard antigen was deposited in the centre of the dipstick and air dried. The unbound sites on the nitrocellulose membrane were blocked by dipping in 5% spray-dried milk powder (Amul, India) in PBST (0.1% Tween 20 in PBS pH 7.4) for 1 h at 37°C. Unbound proteins were removed by washing in PBST solution. Dipsticks were then incubated with the test serum at 37°C for 45 min. Dipsticks were washed four times in PBST, dipped in conjugate and incubated for 30 min at 37°C. Subsequently, they were washed thoroughly and dipped in substrate solution for 2-3 min. The reaction was stopped with PBS pH 7.4. Development of brown color at the site of antigen deposition was considered positive for BTV antibodies. RESULTS
All the animals injected with BTV 1 developed mild clinical signs within 6-9 days. These included dullness, depression, hyperemia of buccal mucosa, gums and lips, and pyrexia. The serological results of the study have been
292
S. C H A N D E R ET 4 L .
TABLE
1
Comparison o f D I A , i n d i r e c t infected with BTV type
tally
.Animal no.
~
3
4
5,6
7
8,9,10
ELISA, SNT
and AGPT for detection of antibody in sheep
Days post infection
Test results DIA
I-ELISA
SNT
AGPT
7
+
+
-
-
14,21,28,43,60
+
+
+
+
0
.
7
+
+
--
-
14, 2 1 , 2 8 , 4 3 , 6 0
+
+
+
+
0
.
7
+
+
-
-
14,21,28,43,60
+
+
+
+
0
.
.
.
.
.
.
.
.
.
.
7,14
+
+
-
+
21,28,43,60
+
+
+
+
0
.
.
.
.
7,14
-
+
-
-
21,28,43,60
+
+
+
+
0
.
.
.
.
7,14
-
+
-
-
21,28,43,60
+
+
+
--
0-60
.
.
.
immunobinding assay; I-ELISA=indirect enzyme-linked neutralization test; A G P T = a g a r gel precipitation test. p o s i t i v e ; - = negative.
DIA=dot
experimen-
1
.
immunosorbent
assay;
SNT=serum + =
summarized in Table 1. Precipitating antibodies as detected by A G P T were first demonstrated at 14 dpi and persisted till the termination of the experiment (60 days). Three animals became positive for BTV antibodies 14 dpi, three animals at 21 dpi, while in one animal precipitating antibodies could not be detected throughout the course of investigation. Neutralizing antibodies were detected after 14 dpi all infected animals. Mean titer of neutralizing antibodies ranged from 10 at 14 dpi to 140 at 2 8 - 6 0 dpi. BTV antibodies in serum of all animals of group 1, were detectable from 7 to 60 dpi by ELISA with mean titers ranging from 1257 at 7 dpi to 5257 at 43 dpi, declining to 4457 at 60 dpi. DIA detected BTV antibodies as early as 7 dpi in four animals while in three other animals D I A could detect antibodies only after 21 dpi. The mean titer of antibodies by D I A varied from 640 at 7 dpi to 3600 at 4 3 60 dpi. DISCUSSION
The most c o m m o n l y used test for BTV detection has been AGPT. This test generally fails to detect BTV antibodies in a considerable number of animals
DIA FOR B L U E T O N G U E V | R U S ANTIBODIES IN SHEEP
293
otherwise positive in the virus isolation test and other serological tests (Stott et al., 1982; Hubschle et al., 1981; Gupta et al., 1990). Recent developments indicate possible replacement of AGPT by enzyme based immunoassays (Manning and Chen, 1980; Poli et al., 1982; Anderson, 1984). In the present investigation, AGPT failed to detect BTV antibodies at early stages of infection, while DIA and ELISA detected BTV antibodies as early as 7 dpi. Similar observations have been made by earlier workers where anti-BTV antibodies were detected as early as 7 dpi by blocking dot-ELISA, while no precipitating antibody could be demonstrated by AGPT (Afshar et al., 1987a, b). In our previous study, the sera tested positive by AGPT were also tested positive by DIA, which indicated the reliability of the test (Gupta et al., 1990). Similarly in the present study, complete agreement was observed when AGPT positive samples were assayed by DIA. When SNT was compared with DIA and ELISA, it was observed that neutralizing antibodies were detectable after 14 dpi while DIA and ELISA were able to detect antibodies at 7 dpi. As compared to DIA, plate ELISA was able to detect BTV antibodies in more sheep at 7 dpi. This may be due to the fact that DIA results were interpreted visually while ELISA results were obtained by an ELISA reader. Similar explanation could be given for more positive results obtained with ELISA as compared to DIA. This discrepancy could, however, be solved by using reflectance densitometry (Towbin and Gordon, 1984 ). Though indirect microplate ELISA is highly specific, rapid and quantitative, it suffers from certain limitations like the need for sophisticated equipment and relatively large quantities of purified antigen to coat microtiter plates. Passive adsorption of certain antigens may be variable for different brands of plates. The aim of DIA is to screen large numbers of serum samples and its utility lies in its simplicity and rapidity in obtaining positive or negative results. The time required in DIA can be shortened by using antigen coated preblocked dip-sticks which can be stored under refrigeration temperature for a long time. As this test does not require any sophistication, it may prove to be a very useful field test in the remote areas where facilities for other tests are not available. ACKNOWLEDGEMENTS
The authors are thankful to Dr. Yogesh Gupta, Shri Chandan Singh, Mangat Ram and Sh. Jit Lal for providing help during this study. The financial support provided by Haryana Agricultural University is gratefully acknowledged. REFERENCES Afshar, A., Thomas, F.C., Wright, P.F., Shapiro, J.L., Anderson, J. and Fulton, R.W., 1987a. Blocking dot ELISA using a monoclonal antibody for detection of antibodies to bluetongue virus in bovine and ovine sera. J. Virol. Methods, 18:271-280.
294
S. C H A N D E R ET AL.
Afshar, A., Thomas, F.C., Wright, P.F., Shapiro, J.L., Shettigara, P.T. and Anderson, J . , 1987b. Comparison of competetive and indirect enzyme linked immunosorbent assays for detection of bluetongue virus antibodies in serum and whole blood. J. Clin. Microbiol., 25: 17051710. Anderson, J., 1984. Use of monoclonal antibody in a blocking ELISA to detect group specific antibodies to bluetongue. J. lmmunol. Methods, 74:139-149. Gupta, Y., 1988. Isolation and serological characterization ofbluetongue virus. M.V.Sc. thesis submitted to Haryana Agricultural University, Hisar, India. Gupta, Y., Chand, P., Singh, A. and Jain, N.C., 1990. Dot immunobindingassay in comparison with enzyme linked immunosorbent assay. Vet. Microbiol., 22:365-371. Hawkes, R., 1986. The dot immunobindingassay. In: J.J. Longone and H.V. Vunakis (Editors), Methods in Enzymology Academic Press Inc., Harcourt Brance, Jovanivich, vol. 121 pp. 484-491. Howell, P.G., 1960. A preliminary antigenic classification of strains of bluetongue virus. Onderstepoort J. Vet. Res., 28: 357-364. Hubschle, O.J.B., Lorenz, R.J. and Matheka, H.D., 1981. Enzyme linked immunosorbent assay for detection of bluetongue virus antibodies. Am. J. Vet. Res., 42:61-65. Jochin, M.M. and Chow, T.L., 1969. Immunodiffusion of bluetongue virus. Am. J. Vet. Res., 30: 33-41. Manning, J.S. and Chen, M.F., 1980. Bluetongue virus: Detection ofantiviral immunoglobulin G by means of enzyme linked immunosorbent assay. Curr. Microbiol., 4: 381-385. Murphy, F.A., Borden, E.C., Shope, R.E. and Harrison, A.K., 1971. Physiological and morphological relationship of some arthropod borne viruses to bluetongue virus, a new toxonomic group, electron microscopic studies. J. Gen. Virol., 13: 273-288. Osburn, B.I., McGowan, B., Heron, G., Ioomis, E., Bushnell, R., Scott, J.L. and Utterback, W., 1981. Epizootiologic study of bluetongue: virologic and serologic results. Am. J. Vet. Res., 42: 884-887. Poli, G., Stott, J.L., Liv, Y.S. and Manning, J.S., 1982. Bluetongue virus: comparative evaluation of enzyme linked immunosorbent assay, immunodiffusionand serum neutralization for detection of viral antibodies. J. Clin. Microbiol., 15:159-162. Stocker, M. and Macpherson, I., 1961. Studies on transformation of hamster cells by polyoma virus in vitro. Virology, 14: 359-370. Stott, J.L., Osburn, B.I. and Barber, T.L., 1982. Recovery of dual serotypes of bluetongue virus from infected sheep and cattle. Vet. Microbiol., 7:197-207. Towbin, H. and Gordon, J., 1984. Immunoblotting and immunobinding: current status and outlook. J. Immunol. Methods, 72:313-340.
289
Dot immunobinding assay for the detection of bluetongue virus antibodies in sheep experimentally inoculated with bluetongue virus type 1 S. Chander, G. Prasad and N.C. Jain Department of Veterinary Microbiology, College Qf Veterinary Sciences, Ha~vana Agricultural University, Hisar-125004, India (Accepted 30 October 1990 )
ABSTRACT Chander, S., Prasad, G. and Jain, N.C., 1991. Dot immunobinding assay for the detection of bluetongue virus antibodies in sheep experimentally inoculated with bluetongue virus type 1. Vet. Microbiol., 28: 289-294. Dot immunobinding assay (D1A) was evaluated for the detection of bluetongue virus (BTV) antibodies in sheep experimentally inoculated with BTV 1. Serum samples collected on 14, 21, 28, 43 and 60 day post infection (dpi) were positive for precipitating antibodies by the agar gel precipitation test (AGPT) while antibodies could be detected as early as 7 dpi by DIA and ELISA. Virus neutralizing antibodies were detected first at 14 dpi. The sensitivity of the four tests was compared on the same serum samples collected at different intervals. The results indicated that DIA was more sensitive than AGPT and the serum neutralization test and as sensitive as ELISA. Thus due to sensitivity simplicity and economy, DIA could replace AGPT for diagnosis and serological survey for BTV infection in animals.
INTRODUCTION
Bluetongue is an insect transmitted viral disease of sheep, goat, cattle and other ruminants. It is caused by bluetongue virus (BTV) which is the prototype orbivirt~s of the family Reoviridae (Murphy et al., 1971 ). Currently the agar gel precipitation test (AGPT) is being used all over the world for screening the animals for BTV antibodies. Reliability of AGPT has been questioned by several workers (Manning and Chen, 1980; Osburn et al., 1981; Stott et al., 1982 ). Enzyme-linked immunosorbent assay (ELISA) has been used during the last decade for detection of group specific BTV antibodies and is comparatively more sensitive than AGPT (Hubschle et al., 1981; Poli et al., 1982; Gupta et al., 1990). Since the ELISA requires sophisticated equipment for
290
s. CHANDER ETAL.
reading the results, dot immunobinding assay (DIA), a modification of ELISA on nitrocellulose membranes, has been developed (Hawkes, 1986; Afshar et al., 1987a; Gupta et al., 1990). In the present study DIA was evaluated for the detection of BTV antibodies in experimentally inoculated sheep. The BTV antibodies were also determined by AGPT, SNT and ELISA for comparative evaluation. MATERIALS AND METHODS
Cell culture Baby hamster kidney (BHK-21 ) cell line, obtained from the Indian Veterinary Research Institute, Mukteshwar, was grown in tissue culture bottles a n d / o r 15 × 125 m m test tubes following the method of Stocker and Macpherson (1961). Growth m e d i u m contained BHK-21 m e d i u m (Glasgow modification) supplemented with 10% inactivated precolostral calf serum and antibodies (penicillin 100 I U / m l , streptomycin sulfate 100 j~g/ml). VirblS
Bluetongue virus serotype 1 available at our laboratory was used to infect the sheep. Animals The 1-year-old crossbred sheep (Nali × Corriedale) free from BTV antibodies were randomly divided in to two groups. Group 1 (seven sheep) was injected with 10 ml of 10 6 TCIDs0/ml of BTV per animal intradermally and subcutaneously at multiple sites. Group 2 (three sheep) were intact, uninfected control sheep. All animals were kept in a midge-proof house. Serum samples Serum samples were collected at 0, 7, 14, 21, 28, 43 and 60 days post infection (dpi) for the detection of BTV antibodies. Serological tests Agar gel precipitation test (AGPT) was carried out following the method of Jochim and Chow ( 1969 ) using standard group specific BTV antigen supplied by Dr. M.M. Jochim, Director, Veterinary Diagnostic Technology Inc. Wheat Ridge, USA. Serum neutralization test (SNT) Neutralizing antibodies against BTV were detected with the method of Howell (1960). The reciprocal of highest serum dilution showing 50% inhibition of cytopathic effect (CPE) was taken as titer of neutralizing antibodies.
DIA FOR BLUETONGUE VIRUS ANTIBODIES IN SHEEP
291
Elisa and dia Substrate. O-phenylenediamine (Sigma, USA) was used as substrate in ELISA, while diaminobenzidine tetrahydrochloride (Sigma, USA) was used in DIA as described by Gupta et al. (1990). Conjugate. Horse radish peroxidase conjugated to rabbit anti-sheep immunoglobulins (DAKOPAT, Denmark) were used at 1:2000 dilution in both tests as described by Gupta et al. (1990). Antigen and antiserum. BTV antigen and antiserum were the same as used for AGPT (Bluetongue antibody test kit, Veterinary Diagnostic Technology, Inc. Wheat Ridge, USA). Indirect ELISA The standard BTV antigen was diluted in carbonate-bicarbonate buffer (pH 9.6) to an OD28o of 0.01 for coating ELISA plates. All other steps of the test were performed as described by Gupta (1988). Absorbance was read at 492 nm wavelength by an ELISA reader (Titertech, Multiscan, Flow Lab.). The end point value of OD was calculated as described by Hubschle et al. ( 1981 ). An OD above 0.3 was taken as positive while an OD equal to or less than 0.3 was considered negative. Dot immunobinding assay Nitrocellulose membranes bound to plastic strips (East Punjab Mfg. Co. Hisar, India) were used for DIA. The test was performed using the protocol developed in our laboratory (Gupta et al., 1990). Briefly, 1 ~tl of the standard antigen was deposited in the centre of the dipstick and air dried. The unbound sites on the nitrocellulose membrane were blocked by dipping in 5% spray-dried milk powder (Amul, India) in PBST (0.1% Tween 20 in PBS pH 7.4) for 1 h at 37°C. Unbound proteins were removed by washing in PBST solution. Dipsticks were then incubated with the test serum at 37°C for 45 min. Dipsticks were washed four times in PBST, dipped in conjugate and incubated for 30 min at 37°C. Subsequently, they were washed thoroughly and dipped in substrate solution for 2-3 min. The reaction was stopped with PBS pH 7.4. Development of brown color at the site of antigen deposition was considered positive for BTV antibodies. RESULTS
All the animals injected with BTV 1 developed mild clinical signs within 6-9 days. These included dullness, depression, hyperemia of buccal mucosa, gums and lips, and pyrexia. The serological results of the study have been
292
S. C H A N D E R ET 4 L .
TABLE
1
Comparison o f D I A , i n d i r e c t infected with BTV type
tally
.Animal no.
~
3
4
5,6
7
8,9,10
ELISA, SNT
and AGPT for detection of antibody in sheep
Days post infection
Test results DIA
I-ELISA
SNT
AGPT
7
+
+
-
-
14,21,28,43,60
+
+
+
+
0
.
7
+
+
--
-
14, 2 1 , 2 8 , 4 3 , 6 0
+
+
+
+
0
.
7
+
+
-
-
14,21,28,43,60
+
+
+
+
0
.
.
.
.
.
.
.
.
.
.
7,14
+
+
-
+
21,28,43,60
+
+
+
+
0
.
.
.
.
7,14
-
+
-
-
21,28,43,60
+
+
+
+
0
.
.
.
.
7,14
-
+
-
-
21,28,43,60
+
+
+
--
0-60
.
.
.
immunobinding assay; I-ELISA=indirect enzyme-linked neutralization test; A G P T = a g a r gel precipitation test. p o s i t i v e ; - = negative.
DIA=dot
experimen-
1
.
immunosorbent
assay;
SNT=serum + =
summarized in Table 1. Precipitating antibodies as detected by A G P T were first demonstrated at 14 dpi and persisted till the termination of the experiment (60 days). Three animals became positive for BTV antibodies 14 dpi, three animals at 21 dpi, while in one animal precipitating antibodies could not be detected throughout the course of investigation. Neutralizing antibodies were detected after 14 dpi all infected animals. Mean titer of neutralizing antibodies ranged from 10 at 14 dpi to 140 at 2 8 - 6 0 dpi. BTV antibodies in serum of all animals of group 1, were detectable from 7 to 60 dpi by ELISA with mean titers ranging from 1257 at 7 dpi to 5257 at 43 dpi, declining to 4457 at 60 dpi. DIA detected BTV antibodies as early as 7 dpi in four animals while in three other animals D I A could detect antibodies only after 21 dpi. The mean titer of antibodies by D I A varied from 640 at 7 dpi to 3600 at 4 3 60 dpi. DISCUSSION
The most c o m m o n l y used test for BTV detection has been AGPT. This test generally fails to detect BTV antibodies in a considerable number of animals
DIA FOR B L U E T O N G U E V | R U S ANTIBODIES IN SHEEP
293
otherwise positive in the virus isolation test and other serological tests (Stott et al., 1982; Hubschle et al., 1981; Gupta et al., 1990). Recent developments indicate possible replacement of AGPT by enzyme based immunoassays (Manning and Chen, 1980; Poli et al., 1982; Anderson, 1984). In the present investigation, AGPT failed to detect BTV antibodies at early stages of infection, while DIA and ELISA detected BTV antibodies as early as 7 dpi. Similar observations have been made by earlier workers where anti-BTV antibodies were detected as early as 7 dpi by blocking dot-ELISA, while no precipitating antibody could be demonstrated by AGPT (Afshar et al., 1987a, b). In our previous study, the sera tested positive by AGPT were also tested positive by DIA, which indicated the reliability of the test (Gupta et al., 1990). Similarly in the present study, complete agreement was observed when AGPT positive samples were assayed by DIA. When SNT was compared with DIA and ELISA, it was observed that neutralizing antibodies were detectable after 14 dpi while DIA and ELISA were able to detect antibodies at 7 dpi. As compared to DIA, plate ELISA was able to detect BTV antibodies in more sheep at 7 dpi. This may be due to the fact that DIA results were interpreted visually while ELISA results were obtained by an ELISA reader. Similar explanation could be given for more positive results obtained with ELISA as compared to DIA. This discrepancy could, however, be solved by using reflectance densitometry (Towbin and Gordon, 1984 ). Though indirect microplate ELISA is highly specific, rapid and quantitative, it suffers from certain limitations like the need for sophisticated equipment and relatively large quantities of purified antigen to coat microtiter plates. Passive adsorption of certain antigens may be variable for different brands of plates. The aim of DIA is to screen large numbers of serum samples and its utility lies in its simplicity and rapidity in obtaining positive or negative results. The time required in DIA can be shortened by using antigen coated preblocked dip-sticks which can be stored under refrigeration temperature for a long time. As this test does not require any sophistication, it may prove to be a very useful field test in the remote areas where facilities for other tests are not available. ACKNOWLEDGEMENTS
The authors are thankful to Dr. Yogesh Gupta, Shri Chandan Singh, Mangat Ram and Sh. Jit Lal for providing help during this study. The financial support provided by Haryana Agricultural University is gratefully acknowledged. REFERENCES Afshar, A., Thomas, F.C., Wright, P.F., Shapiro, J.L., Anderson, J. and Fulton, R.W., 1987a. Blocking dot ELISA using a monoclonal antibody for detection of antibodies to bluetongue virus in bovine and ovine sera. J. Virol. Methods, 18:271-280.
294
S. C H A N D E R ET AL.
Afshar, A., Thomas, F.C., Wright, P.F., Shapiro, J.L., Shettigara, P.T. and Anderson, J . , 1987b. Comparison of competetive and indirect enzyme linked immunosorbent assays for detection of bluetongue virus antibodies in serum and whole blood. J. Clin. Microbiol., 25: 17051710. Anderson, J., 1984. Use of monoclonal antibody in a blocking ELISA to detect group specific antibodies to bluetongue. J. lmmunol. Methods, 74:139-149. Gupta, Y., 1988. Isolation and serological characterization ofbluetongue virus. M.V.Sc. thesis submitted to Haryana Agricultural University, Hisar, India. Gupta, Y., Chand, P., Singh, A. and Jain, N.C., 1990. Dot immunobindingassay in comparison with enzyme linked immunosorbent assay. Vet. Microbiol., 22:365-371. Hawkes, R., 1986. The dot immunobindingassay. In: J.J. Longone and H.V. Vunakis (Editors), Methods in Enzymology Academic Press Inc., Harcourt Brance, Jovanivich, vol. 121 pp. 484-491. Howell, P.G., 1960. A preliminary antigenic classification of strains of bluetongue virus. Onderstepoort J. Vet. Res., 28: 357-364. Hubschle, O.J.B., Lorenz, R.J. and Matheka, H.D., 1981. Enzyme linked immunosorbent assay for detection of bluetongue virus antibodies. Am. J. Vet. Res., 42:61-65. Jochin, M.M. and Chow, T.L., 1969. Immunodiffusion of bluetongue virus. Am. J. Vet. Res., 30: 33-41. Manning, J.S. and Chen, M.F., 1980. Bluetongue virus: Detection ofantiviral immunoglobulin G by means of enzyme linked immunosorbent assay. Curr. Microbiol., 4: 381-385. Murphy, F.A., Borden, E.C., Shope, R.E. and Harrison, A.K., 1971. Physiological and morphological relationship of some arthropod borne viruses to bluetongue virus, a new toxonomic group, electron microscopic studies. J. Gen. Virol., 13: 273-288. Osburn, B.I., McGowan, B., Heron, G., Ioomis, E., Bushnell, R., Scott, J.L. and Utterback, W., 1981. Epizootiologic study of bluetongue: virologic and serologic results. Am. J. Vet. Res., 42: 884-887. Poli, G., Stott, J.L., Liv, Y.S. and Manning, J.S., 1982. Bluetongue virus: comparative evaluation of enzyme linked immunosorbent assay, immunodiffusionand serum neutralization for detection of viral antibodies. J. Clin. Microbiol., 15:159-162. Stocker, M. and Macpherson, I., 1961. Studies on transformation of hamster cells by polyoma virus in vitro. Virology, 14: 359-370. Stott, J.L., Osburn, B.I. and Barber, T.L., 1982. Recovery of dual serotypes of bluetongue virus from infected sheep and cattle. Vet. Microbiol., 7:197-207. Towbin, H. and Gordon, J., 1984. Immunoblotting and immunobinding: current status and outlook. J. Immunol. Methods, 72:313-340.
